Voltage-dependent ion channels form aqueous pores in membranes that open and close in response to changes in transmembrane potential. Although many channels of this kind have been the subject of intense molecular and biophysical investigation, a true molecular picture of how voltage sensor movement is coupled to channel opening and the precise nature of the underlying protein conformational changes remain poorly understood despite much speculation. The long term goal of this proposal is to use the outer mitochondrial membrane channel VDAC as a model of how a voltage "sensor" couples to other regions of a protein to produce the molecular rearrangements generating channel gating. Over the past period of funding, we have examined the functional consequences of a large number of charge changes introduced by site-directed mutagenesis throughout the yeast VDAC (YVDAC) molecule. These functional studies have led to a model of the transmembrane topology of the yeast VDAC protein in the open state, defined regions of the protein that are removed from the pore during channel closure and identified specific residues forming the voltage sensor. In addition, human VDAC (HVDAC) genes have been identified and characterized. Over the course of the next funding period, we will build on these studies by first testing the hypothesis that a similar overall transmembrane topology of VDAC proteins generates VDAC's highly conserved physiology. To test this hypothesis, we will examine the folding pattern of HVDAC 1 by functional studies of mutant HVDAC 1 proteins. HVDAC 1 has a very different primary sequence yet identical physiological properties. These studies should allow us to test the generality of models developed in our studies of YVDAC. Second, we will test our models of the topology of the open VDAC channel and the conformational changes accompanying voltage-gating at the protein level by site-specific biotinylation of single cysteine residues (cys). Single cys residues will be engineered into the yeast protein and modified with thiol specific reagents that attach a biotin at this site. The accessibility of the biotin-modified cys to streptavidin (strp) and the location of the strp if bound will be used to localize this residue with respect to the membrane in a variety of protein conformations both in the outer membranes and in bilayers. Third, we will begin to test the idea that VDAC's channel properties in the cell are regulated by association with cytoplasmic and intermitochondrial membrane space proteins co-ordinated into a complex with VDAC. By use of the yeast two hybrid system, we have identified three novel molecules which interact strongly with yeast VDAC. Our goals are to characterize the proteins encoded by these and other genes identified in our screens, convincingly demonstrate their interaction with VDAC either in vitro or in vivo, and determine their potential effect in modulating VDAC's channel characteristics in planar phospholipid bilayers.